Self-replicating RNA molecule from hepatitis C virus

ABSTRACT

A unique HCV RNA molecule is provided having an enhanced efficiency of establishing cell culture replication. Novel adaptive mutations have been identified within the HCV non-structural region that improves the efficiency of establishing persistently replicating HCV RNA in cell culture. This self-replicating polynucleotide molecule contains, contrary to all previous reports, a 5′-NTR that can be either an A as an alternative to the G already disclosed and therefore provides an alternative to existing systems comprising a self-replicating HCV RNA molecule. The G→A mutation gives rise to HCV RNA molecules that, in conjunction with mutations in the HCV non-structural region, such as the G(2042)C/R mutations, possess greater efficiency of transduction and/or replication. These RNA molecules when transfected in a cell line are useful for evaluating potential inhibitors of HCV replication.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/686,835, filed Oct. 16, 2003, which is a continuation of U.S.application Ser. No. 10/029,907, filed Dec. 21, 2001, which claims, asdoes the present application priority to U.S. Provisional ApplicationSer. No. 60/257,857 filed on Dec. 22, 2000, the disclosures of all ofwhich are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to a HCV RNA molecule thatself-replicates in appropriate cell lines, particularly to aself-replicating HCV RNA construct having an enhanced efficiency ofestablishing cell culture replication.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is the major etiological agent ofpost-transfusion and community-acquired non-A non-B hepatitis worldwide.It is estimated that over 200 million people worldwide are infected bythe virus. A high percentage of carriers become chronically infected andmany progress to chronic liver disease, so called chronic hepatitis C.This group is in turn at high risk for serious liver disease such asliver cirrhosis, hepatocellular carcinoma and terminal liver diseaseleading to death. The mechanism by which HCV establishes viralpersistence and causes a high rate of chronic liver disease has not beenthoroughly elucidated. It is not known how HCV interacts with and evadesthe host immune system. In addition, the roles of cellular and humoralimmune responses in protection against HCV infection and disease haveyet to be established.

Various clinical studies have been conducted with the goal ofidentifying pharmaceutical compounds capable of effectively treating HCVinfection in patients afflicted with chronic hepatitis C. These studieshave involved the use of interferon-alpha, alone and in combination withother antiviral agents such as ribavirin. Such studies have shown that asubstantial number of the participants do not respond to thesetherapies, and of those that do respond favorably, a large proportionwere found to relapse after termination of treatment. To date there areno broadly effective antiviral compounds for treatment of HCV infection.

HCV is an enveloped positive strand RNA virus in the Flaviviridaefamily. The single strand HCV RNA genome is of positive polarity andcomprises one open reading frame (ORF) of approximately 9600 nucleotidesin length, which encodes a linear polyprotein of approx. 3010 aminoacids. In infected cells, this polyprotein is cleaved at multiple sitesby cellular and viral proteases to produce structural and non-structural(NS) proteins. The structural proteins (C, E1, E2 and E2-p7) comprisepolypeptides that constitute the virus particle (Hijikata, M. et al.,1991, Proc. Natl. Acad. Sci. USA. 88, 5547-5551; Grakoui et al.,1993(a), J. Virol. 67, 1385-1395). The non-structural proteins (NS2,NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factorsthat catalyze and regulate the replication of the HCV RNA genome.Processing of the structural proteins is catalyzed by host cellproteases (Hijikata, M. et al., 1991, Proc. Natl. Acad. Sci. USA. 88,5547-5551). The generation of the mature non-structural proteins iscatalyzed by two virally encoded proteases. The first is the NS2/3zinc-dependent metalloprotease which auto-catalyses the release of theNS3 protein from the polyprotein. The released NS3 contains a N-terminalserine protease domain (Grakoui et al., 1993(b), Proc Natl Acad Sci USA,90, 10583-7; Hijikata, M. et al., 1993, J. Virol. 67, 4665-4675) andcatalyzes the remaining cleavages from the polyprotein. The releasedNS4A protein has at least two roles. First, forming a stable complexwith NS3 protein and assisting in the membrane localization of theNS3/NS4A complex (Kim et al., Arch Virol. 1999, 144, 329-343) andsecond, acting as a cofactor for NS3 protease activity. Thismembrane-associated complex, in turn catalyzes the cleavage of theremaining sites on the polyprotein, thus effecting the release of NS4B,NS5A and NS5B (Bartenschlager, R. et al., 1993, J. Virol., 67,3835-3844; Grakoui et al., 1993(a), J. Virol. 67, 1385-1395; Hijikata,M. et al., 1993, J. Virol. 67, 4665-4675; Love, R. A. et al., 1996,Cell, 87, 331-342; reviewed in Kwong et al., 1998 Antiviral Res., 40,1-18). The C-terminal segment of the NS3 protein also harbors nucleosidetriphosphatase and RNA helicase activity (Kim et al., 1995, Biochem.Biophys. Res. Comm., 215, 160-166.). The function of the protein NS4B isunknown. NS5A, a highly phosphorylated protein, seems to be responsiblefor the Interferon resistance of various HCV genotypes (Gale Jr. et al.1997 Virology 230, 217; Reed et al., 1997 J. Virol. 71, 7187. NS5B is anRNA-dependent RNA polymerase (RdRp) that is involved in the replicationof HCV.

The open reading frame of the HCV RNA genome is flanked on its 5′ end bya non-translated region (NTR) of approx. 340 nucleotides that functionsas the internal ribosome entry site (IRES), and on its 3′ end by a NTRof approximately 230 nucleotides. Both the 5′ and 3′ NTRs are importantfor RNA genome replication. The genomic sequence variance is not evenlydistributed over the genome and the 5′NTR and parts of the 3′NTR are themost highly conserved portions. The authentic, highly conserved 3′NTR isthe object of U.S. Pat. No. 5,874,565 granted to Rice et al.

The cloned and characterized partial and complete sequences of the HCVgenome have also been analyzed with regard to appropriate targets for aprospective antiviral therapy. Four viral enzyme activities providepossible targets such as (1) the NS2/3 protease; (2) the NS3/4A proteasecomplex, (3) the NS3 Helicase and (4) the NS5B RNA-dependent RNApolymerase. The NS3/4A protease complex and the NS3 helicase havealready been crystallized and their three-dimensional structuredetermined (Kim et al., 1996, Cell, 87, 343; Yem et al. Protein Science,7, 837, 1998; Love, R. A. et al., 1996, Cell, 87, 331-342; Kim et al.,1998, Structure, 6, 89; Yao et al., 1997 Nature Structural Biology, 4,463; Cho et al., 1998, J. Biol. Chem., 273, 15045). The NS5B RNAdependent RNA polymerase has also been crystallized to reveal astructure reminiscent of other nucleic acid polymerases (Bressanelli etal. 1999, Proc. Natl. Acad. Sci, USA 96, 13034-13039; Ago et al. 1999,Structure 7, 1417-1426; Lesburg et al. 1999, Nat. Struct. Biol. 6,937-943).

Even though important targets for the development of a therapy forchronic HCV infection have been defined with these enzymes and eventhough a worldwide intensive search for suitable inhibitors is ongoingwith the aid of rational drug design and HTS, the development of therapyhas one major deficiency, namely the lack of cell culture systems orsimple animal models, which allow direct and reliable propagation of HCVviruses. The lack of an efficient cell culture system is still the mainreason to date that an understanding of HCV replication remains elusive.

Although flavi- and pestivirus self-replicating RNAs have been describedand used for the replication in different cell lines with a relativelyhigh yield, similar experiments with HCV have not been successful todate (Khromykh et al., 1997, J. Virol. 71, 1497; Behrens et al., 1998,J. Virol. 72, 2364; Moser et al., 1998 J. Virol. 72, 5318). It is knownfrom different publications that cell lines or primary cell cultures canbe infected with high-titer patient serum containing HCV (Lanford et al.1994 Virology 202, 606; Shimizu et al. 1993 PNAS, USA 90, 6037-6041;Mizutani et al. 1996 J. Virol. 70, 7219-7223; Ikda, et al. 1998, VirusRes. 56, 157; Fourner et al. 1998, J. Gen. Virol. 79, 2376; Ito et al.1996, J. Gen. Virol. 77, 1043-1054). However, these virus-infected celllines or cell cultures do not allow the direct detection of HCV-RNA orHCV antigens.

It is also known from the publications of Yoo et al. 1995 J. Virol., 69,32-38; and of Dash et al., 1997, Am. J. Pathol., 151, 363-373; thathepatoma cell lines can be transfected with synthetic HCV-RNA obtainedthrough in vitro transcription of the cloned HCV genome. In bothpublications the authors started from the basic idea that the viral HCVgenome is a plus-strand RNA functioning directly as mRNA after beingtransfected into the cell, permitting the synthesis of viral proteins inthe course of the translation process, and so new HCV particles couldform HCV viruses and their RNA detected through RT-PCR. However thepublished results of the RT-PCR experiments indicate that the HCVreplication in the described HCV transfected hepatoma cells is notparticularly efficient and not sufficient to measure the quality ofreplication, let alone measure the modulations in replication afterexposure to potential antiviral drugs. Furthermore it is now known thatthe highly conserved 3′ NTR is essential for the virus replication(Yanagi et al., 1999 Proc. Natl. Acad. Sci. USA, 96, 2291-95). Thisknowledge strictly contradicts the statements of Yoo et al. J. Virol.,69, 32-38(supra) and Dash et al., 1997, Am. J. Pathol., 151, 363-373.(supra), who used for their experiments only HCV genomes with shorter 3′NTRs and not the authentic 3′ end of the HCV genome.

In WO 98/39031, Rice et al. disclosed authentic HCV genome RNAsequences, in particular containing: a) the highly conserved 5′-terminalsequence “GCCAGCC” (SEQ ID NO. 26); b) the HCV polyprotein codingregion; and c) 3′-NTR authentic sequences.

In WO 99/04008, Purcell et al. disclosed an HCV infectious clone thatalso contained only the highly conserved 5′-terminal sequence “GCCAGC”(SEQ ID NO. 27).

Recently Lohman et al. 1999 (Science 285, 110-113) and Bartenschlager,R. et al., 1993, J. Virol., 67, 3835-3844(in CA 2,303,526, laid-open onOct. 3, 2000) disclosed a HCV cell culture system where the viral RNA(I377/NS2-3′) self-replicates in the transfected cells with suchefficiency that the quality of replication can be measured with accuracyand reproducibility. The Lohman and Bartenschlager, R. et al., 1993, J.Virol., 67, 3835-3844 disclosures were the first demonstration of HCVRNA replication in cell culture that was substantiated through directmeasurement by Northern blots. This replicon system and sequencesdisclosed therein highlight once again the conserved 5′ sequence“GCCAGC” (SEQ ID NO. 27). A similar observation highlighting theconservation of the 5′NTR was made by Blight et al. 2000 (Science 290,1972-1974) and WO 01/89364 published on Nov. 29, 2001.

In addition to the conservation of the 5′ and 3′ untranslated regions incell culture replicating RNAs, three other publications by Lohman et al.2001, J. Virol. 1437-1449 Krieger et al. 2001 J. Virol. 4614-4624 andGuo et al., (2001) J. Virol. 8516-8523 have recently disclosed distinctadaptive mutants within the HCV non-structural protein coding region.Specific nucleotide changes that alter the amino acids of the HCVnon-structural proteins are shown to enhance the efficiency ofestablishing stable replicating HCV subgenomic replicons in culturecells.

Applicant has now found that, contrary to all previous reports, thehighly conserved 5′-NTR can be mutated by adaptation to give rise to aHCV RNA sequence that, in conjunction with mutations in the HCVnon-structural region, provides for a greater efficiency of transductionand/or replication.

Applicant has also identified novel adaptive mutations within the HCVnon-structural region that improves the efficiency of establishingpersistently replicating HCV RNA in cell culture.

One advantage of the present invention is to provide an alternative tothese existing systems comprising a HCV RNA molecule thatself-replicates. Moreover, the present invention demonstrates that theinitiating nucleotide of the plus-strand genome can be either an A as analternative to the G already disclosed.

A further advantage of the present invention is to provide a unique HCVRNA molecule that transduces and/or replicates with higher efficiency.The Applicant demonstrates the utility of this specific RNA molecule ina cell line and its use in evaluating a specific inhibitor of HCVreplication.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a 5′-nontranslated region of the hepatitis C virus wherein its highly conservedguanine at position 1 is substituted for adenine.

Particularly, the present invention provides a hepatitis C viruspolynucleotide comprising adenine at position 1 as numbered according tothe I377/NS2-3′ construct (Lohmann et al. 1999, Science 285, 110-113,Accession # AJ242651).

Particularly, the invention provides a HCV self-replicatingpolynucleotide comprising a 5′-terminus consisting of ACCAGC (SEQ ID NO.8).

In a second embodiment, the present invention is directed to a HCVself-replicating polynucleotide encoding a polyprotein comprising one ormore amino acid substitution selected from the group consisting of:R(1135)K; S(1148)G; S(1560)G; K(1691)R; L(1701)F; I(1984)V; T(1993)A;G(2042)C; G(2042)R; S(2404)P; L(2155)P; P(2166)L and M(2992)T.

Particularly, the invention is directed to a HCV self-replicatingpolynucleotide encoding a polyprotein comprising the any one of theamino acid substitutions as described above, further comprising theamino acid substitution E(1202)G.

More particularly, the invention provides a HCV self-replicatingpolynucleotide encoding a polyprotein comprising a G2042C or a G2042Rmutation.

Most particularly, the invention provides for HCV self-replicatingpolynucleotide comprising a nucleotide substitution G→A at position 1,and said polynucleotide encodes a polyprotein further comprising aG2042C or a G2042R mutation.

Particularly, the polynucleotide of the present invention can be in theform of RNA or DNA that can be transcribed to RNA.

In a third embodiment, the invention also provides for an expressionvector comprising a DNA form of the above polynucleotide, operablylinked with a promoter.

According to a fourth embodiment, there is provided a host celltransfected with the self-replicating polynucleotide or the vector asdescribed above.

In a fifth embodiment, the present invention provides a RNA replicationassay comprising the steps of:

-   -   incubating the host cell as described above in the absence or        presence of a potential hepatitis C virus inhibitor;    -   isolating the total cellular RNA from the cells;    -   analyzing the RNA so as to measure the amount of HCV RNA        replicated;    -   comparing the levels of HCV RNA in cells in the absence and        presence of the inhibitor.

In a sixth embodiment, the invention is directed to a method for testinga compound for inhibiting HCV replication, including the steps of:

-   -   a) treating the above described host cell with the compound;    -   b) evaluating the treated host cell for reduced replication,        wherein reduced replication indicates the ability of the        compound to inhibit replication.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the bi-cistronic replicon RNA. Thesequence deviations between the I377/NS2-3′ replicon from Lohman et al.,1999 Science 285: 110-113 and the APGK12 replicon are indicated belowthe replicon. In place of a G nucleotide at the +1 position in theI377/NS2-3′ replicon, the APGK12 contains an additional G resulting inGG at the 5′ terminus (the first G being counted as position −1). In thelinker region between the neo gene and the EMCV IRES sequence two areasdeviate from I377/NS2-3′:14 nucleotides (CGCGCCCAGATGTT) (SEQ ID NO. 28)which are not present in I377/NS2/3 are inserted at position 1184 inAPGK12; 11 nucleotides (1231-1241) present in I377/NS2-3′ are deleted togenerate APGK-12. In the NS5B coding region, a T at position 8032 wasmutated to C to eliminate a NcoI restriction site.

FIG. 2 shows Northern blots of RNA-transfected Huh-7 cell lines. 12 μgof total cellular RNA or control RNA was separated on 0.5%agarose-formaldehyde gels and transferred to Hybond N+ paper, fixed and(FIG. 2A) radioactively probed with HCV specific minus-strand RNA thatdetects the presence of plus-strand replicon RNA. Lanes 1 and 2:positive controls that contain 10⁹ copies of in vitro transcribed APGK12RNA. Lane 3: negative control of total cellular RNA from untransfectedHuh-7 cells. Lanes 4 and 5: cellular RNA from B1 and B3 cell lines thathave integrated DNA copies of the neomycin phosphotransferase gene. Lane6: total cellular RNA from a Huh-7 cell line, designated S22.3, thatharbors high copy number HCV sub-genomic replicon RNA as highlighted bythe arrow. Other cell lines have no detectable replicon RNA. FIG. 2B isidentical to FIG. 2A with the exception that the blot was radioactivelyprobed with HCV specific plus-strand RNA to detect the presence of HCVminus-strand RNA. Lanes 1 and 2 are positive control lanes that contain10⁹ copies of full length HCV minus strand RNA. Lane 6, which contains12 μg of total cellular RNA from cell line S22.3, harbors detectableminus-strand replicon RNA at the expected size of 8-9 kilobases. Mrepresent the migration of non-radioactive molecular size markers on theagarose gel. 28s represents the migration of 28s ribosomal RNA andaccounts for the detection of this species in a samples of totalcellular RNA.

FIG. 3 shows indirect immunofluorescence of a HCV non-structural proteinin the S22.3 cell line. Indirect immunofluorescence was performed oncells that were cultured and fixed, permeabilized and exposed to arabbit polyclonal antibody specific for a segment of the HCV NS4Aprotein. Secondary goat anti-rabbit antibody conjugated with red-fluorAlexa 594 (Molecular Probes) was used for detection. Top panels showsthe results of immunofluorescence (40× objective) and the specificstaining of the S22.3 cells. The bottom panels represent the identicalfield of cells viewed by diffractive interference contrast (DIC)microscopy. The majority of S22.3 (FIG. 3A) cells within the field stainpositively for HCV NS4A protein that localizes in the cytoplasm, whereasthe B1 cells (FIG. 3B) that fail to express any HCV proteins, only havebackground level of staining.

FIG. 4 shows Western-blots following SDS-PAGE separation of totalproteins extracted from three cell lines: (i) naïve Huh-7 cell line,(ii) neomycin resistant Huh-7 cell line B1, and (iii) the S22.3 cellline. Panels A, B, and C, demonstrate the results of western blotsprobed with rabbit polyclonal antisera specific for neomycinphosphotransferase (NPT), HCV NS3, and HCV NS5B, respectively.Visualization was achieved through autoradiographic detection of achemiluminescent reactive secondary goat anti-rabbit antibody. Panel Ashows that the S22.3 RNA replicon cell line, expresses the NPT proteinat levels higher than control B1 cells and that the naïve Huh-7 cellline does not produce the NPT protein. Panels B and C show that only theS22.3 cell line produces the mature HCV NS3 and NS5B proteins,respectively. M represents molecular weight (in kilodaltons) ofpre-stained polypeptide markers.

FIGS. 5A and 5B identify the nucleotide and amino acid sequencesrespectively that differ from the APGK12 sequence in the different HCVbi-cistronic replicons. The S22.3 adapted replicon is a first generationreplicon selected following the transfection of RNA transcribed from theAPGK12 template. R3, R7, R16 are second generation replicons that wereselected following the transfection of RNA isolated from the S22.3 firstgeneration replicon cell line. FIG. 5A: Nucleotide mutations that werecharacterized in each of the adapted replicons are indicated adjacent tothe respective segment of the replicon (IRES, NS3, NS4A, NS5A, andNS5B). FIG. 5B: Amino acid numbers are numbered according to the fulllength HCV poly-protein with the first amino acid in the second cistroncorresponding to amino acid 810 in NS2 of I377/NS2-3′ construct.

FIG. 6 depicts the colony formation efficiency of four in vitrotranscribed HCV sub-genomic bi-cistronic replicon RNAs. The APGK12serves as the reference sequence; highlighted are the initiatingnucleotides of the HCV IRES in each of the constructs and the amino aciddifferences (from the APGK12 reference sequence) in the HCVnon-structural region for the two R3-rep. Note that the in vitrotranscribed APGK-12 RNAs that harbor either a 5′G or 5′A form colonieswith the same efficiency (ca. 80 cfu/μg in panels A and B) followingselection with 0.25 mg/ml G418. RNA isolated from the second generationR3 cell line was reverse transcribed into DNA and cloned into thepAPGK12 vector backbone to generate the R3-rep, which was sequenced andfound to encode additional changes that included the L(2155)Psubstitution in the NS5A segment of the HCV polyprotein (compare R3-repsequence with the R3 sequence in tables 2 and 3). Various quantities ofin vitro transcribed R3-rep-5′A RNA, were transfected into naïve Huh-7cells to determine a colony formation efficiency of 1.2×10⁶ cfu/μg ofRNA (panel C). Various quantities of R3-rep-5′G were also transfectedresulting in a colony formation efficiency of 2×10⁶ cfu/μg of RNA (panelD).

FIG. 7 displays a typical RT-PCR amplification plot (left panel) and thegraphical representation of Ct values versus known HCV RNA quantity in astandard curve (right panel). Each of the plotted curves in the leftpanel, graph the increment of fluorescence reporter signal (delta-Rn)versus PCR cycle number for a predetermined quantity of HCV repliconRNA. The Ct value is obtained by determining the point at which thefluorescence exceeds an arbitrary value (horizontal line). The rightpanel demonstrates the linear relationship between starting RNA copynumber of the predetermined standards (large black dots) and the Ctvalue. Smaller dots are the Ct values of RNA samples (containing unknownquantity of HCV replicon RNA) from S22.3 cells treated with variousconcentrations of a specific inhibitor of HCV replication.

FIG. 8 shows the effect of increasing concentration of inhibitor A onHCV RNA replicon levels in Huh7 cells. S22.3 cells were grown in thepresence of increasing concentrations of inhibitor A starting at 0.5 nMand ranging to 1024 nM. The inhibitor dose-response curve is the resultof 11 concentrations from serial two-fold dilutions (1:1). One controlwell, without any inhibitor, was also included during the course of theexperiment. The cells were incubated for 4 days in a 5% CO₂ incubator at37° C. Total cellular RNA was extracted, quantified by optical density.HCV replicon RNA was evaluated by real time RT-PCR and plotted as genomeequivalents/μg total RNA as a function of inhibitor concentration.

DEFINITIONS

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Generally,the procedures for cell culture, infection, molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as for exampleSambrook et al. (1989) Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Labs and Ausubel et al. (1994).

Nucleotide sequences are presented herein by single strand, in the 5′ to3′ direction, from left to right, using the one letter nucleotidesymbols as commonly used in the art and in accordance with therecommendations of the IUPAC-IUB Biochemical Nomenclature Commission(1972) Biochemistry, 11, 1726-1732.

The present description refers to a number of routinely used recombinantDNA (rDNA) technology terms. Nevertheless, definitions of selectedexamples of such rDNA terms are provided for clarity and consistency.

The term “DNA segment or molecule or sequence”, is used herein, to referto molecules comprised of the deoxyribonucleotides adenine (A), guanine(G), thymine (T) and/or cytosine (C). These segments, molecules orsequences can be found in nature or synthetically derived. When read inaccordance with the genetic code, these sequences can encode a linearstretch or sequence of amino acids which can be referred to as apolypeptide, protein, protein fragment and the like.

As used herein, the term “gene” is well known in the art and relates toa nucleic acid sequence defining a single protein or polypeptide. Thepolypeptide can be encoded by a full-length sequence or any portion ofthe coding sequence, so long as the functional activity of the proteinis retained.

A “structural gene” defines a DNA sequence which is transcribed into RNAand translated into a protein having a specific structural function thatconstitute the viral particles. “Structural proteins” defines the HCVproteins incorporated into the virus particles namely, core “C”, E1, E2,and E2-p7.

“Non-structural proteins”, defines the HCV proteins that are notcomprised in viral particles namely, NS2, NS3, NS4A, NS5A and NS5B.

“Restriction endonuclease or restriction enzyme” is an enzyme that hasthe capacity to recognize a specific base sequence (usually 4, 5 or 6base pairs in length) in a DNA molecule, and to cleave the DNA moleculeat every place where this sequence appears. An example of such an enzymeis EcoRI, which recognizes the base sequence G↓AATTC (SEQ ID NO. 29) andcleaves a DNA molecule at this recognition site.

“Restriction fragments” are DNA molecules produced by the digestion ofDNA with a restriction endonuclease. Any given genome or DNA segment canbe digested by a particular restriction endonuclease into at least twodiscrete molecules of restriction fragments.

“Agarose gel electrophoresis” is an analytical method for fractionatingpolynucleotide molecules based on their size. The method is based on thefact that nucleic acid molecules migrate through a gel as through asieve, whereby the smallest molecule has the greatest mobility andtravels the farthest through the gel. The sieving characteristics of thegel retards the largest molecules such that, these have the leastmobility. The fractionated polynucleotides can be visualized by stainingthe gel using methods well known in the art, nucleic acid hybridizationor by tagging the fractionated molecules with a detectable label. Allthese methods are well known in the art, specific methods can be foundin Ausubel et al. (supra).

“Oligonucleotide or oligomer” is a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three. Theexact size of the molecule will depend on many factors, which in turndepend on the ultimate function or use of the oligonucleotide. Anoligonucleotide can be derived synthetically, by cloning or byamplification.

“Sequence amplification” is a method for generating large amounts of atarget sequence. In general, one or more amplification primers areannealed to a nucleic acid sequence. Using appropriate enzymes,sequences found adjacent to, or in between the primers are amplified. Anamplification method used herein is the polymerase chain reaction (PCR)and can be used in conjunction with the reverse-transcriptase (RT) toproduce amplified DNA copies of specific RNA sequences.

“Amplification primer” refers to an oligonucleotide, capable ofannealing to a RNA or DNA region adjacent to a target sequence andserving as the initiation primer for DNA synthesis under suitableconditions well known in the art. The synthesized primer extensionproduct is complementary to the target sequence.

The term “domain” or “region” refers to a specific amino acid sequencethat defines either a specific function or structure within a protein.As an example herein, is the NS3 protease domain comprised within theHCV non-structural polyprotein.

The terms “plasmid” “vector” or “DNA construct” are commonly known inthe art and refer to any genetic element, including, but not limited to,plasmid DNA, phage DNA, viral DNA and the like which can incorporate theoligonucleotide sequences, or sequences of the present invention andserve as DNA vehicle into which DNA of the present invention can becloned. Numerous types of vectors exist and are well known in the art.

The terminology “expression vector” defines a vector as described abovebut designed to enable the expression of an inserted sequence followingtransformation or transfection into a host. The cloned gene (insertedsequence) is usually placed under the control of control elementsequences such as promoter sequences. Such expression control sequenceswill vary depending on whether the vector is designed to express theoperably linked gene in vitro or in vivo in a prokaryotic or eukaryotichost or both (shuttle vectors) and can additionally containtranscriptional elements such as enhancer elements, terminationsequences, tissue-specificity elements, and/or translational initiationand termination sites.

A host cell or indicator cell has been “transfected” by exogenous orheterologous DNA (e.g. a DNA construct) or RNA, when such nucleic acidhas been introduced inside the cell. The transfecting DNA may or may notbe integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transfecting/transforming DNA may be maintained on anepisomal element such as a plasmid. With respect to eukaryotic cells, anexample of a stably transfected cell is one in which the transfectingDNA has become integrated into a chromosome and is inherited by daughtercells through chromosome replication. A host cell or indicator cell canbe transfected with RNA. A cell can be stably transfected with RNA ifthe RNA replicates and copies of the RNA segregate to daughter cellsupon cell division. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transfecting DNA or RNA.Transfection methods are well known in the art (Sambrook et al., 1989,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Labs; Ausubelet al., 1994, Current Protocols in Molecular Biology, Wiley, N.Y.). Ifthe RNA encodes for a genetic marker that imparts an observablephenotype, such as antibiotic resistance, then the stable transfectionof replicating RNA can be monitored by the acquisition of such phenotypeby the host cell.

As used herein the term “transduction” refers to the transfer of agenetic marker to host cells by the stable transfection of a replicatingRNA.

The nucleotide sequences and polypeptides useful to practice theinvention include without being limited thereto, mutants, homologs,subtypes, quasi-species, alleles, and the like. It is understood thatgenerally, the sequences of the present invention encode a polyprotein.It will be clear to a person skilled in the art that the polyprotein ofthe present invention and any variant, derivative or fragment thereof,is auto-processed to an active protease.

As used herein, the designation “variant” denotes in the context of thisinvention a sequence whether a nucleic acid or amino acid, a moleculethat retains a biological activity (either functional or structural)that is substantially similar to that of the original sequence. Thisvariant may be from the same or different species and may be a naturalvariant or be prepared synthetically. Such variants include amino acidsequences having substitutions, deletions, or additions of one or moreamino acids, provided the biological activity of the protein isconserved. The same applies to variants of nucleic acid sequences whichcan have substitutions, deletions, or additions of one or morenucleotides, provided that the biological activity of the sequence isgenerally maintained.

The term “derivative” is intended to include any of the above describedvariants when comprising additional chemical moiety not normally a partof these molecules. These chemical moieties can have varying purposesincluding, improving a molecule's solubility, absorption, biologicalhalf life, decreasing toxicity and eliminating or decreasing undesirableside effects. Furthermore, these moieties can be used for the purpose oflabeling, binding, or they may be comprised in fusion product(s).Different moieties capable of mediating the above described effects canbe found in Remington's The Science and Practice of Pharmacy (1995).Methodologies for coupling such moieties to a molecule are well known inthe art.

The term “fragment” refers to any segment of an identified DNA, RNA oramino acid sequence and/or any segment of any of the variants orderivatives described herein above that substantially retains itsbiological activity (functional or structural) as required by thepresent invention.

The terms “variant”, “derivative”, and “fragment” of the presentinvention refer herein to proteins or nucleic acid molecules which canbe isolated/purified, synthesized chemically or produced throughrecombinant DNA technology. All these methods are well known in the art.As exemplified herein below, the nucleotide sequences and polypeptidesused in the present invention can be modified, for example by in vitromutagenesis.

As used herein, the term “HCV polyprotein coding region” means theportion of a hepatitis C virus that codes for the polyprotein openreading frame (ORF). This ORF may encode proteins that are the same ordifferent than wild-type HCV proteins. The ORF may also encode only someof the functional protein encoded by wild-type polyprotein codingregion. The protein encoded therein may also be from different isolatesof HCV, and non-HCV protein may also be encoded therein.

As used herein, the abbreviation “NTR” used in the context of apolynucleotide molecule means a non-translated region. The term “UTR”means untranslated region. Both are used interchangeably.

PREFERRED EMBODIMENTS

Particularly, the invention provides a HCV self-replicatingpolynucleotide molecule comprising a 5′-terminus consisting of ACCAGC(SEQ ID NO.8).

According to the first embodiment of this invention, there isparticularly provided a HCV polynucleotide construct comprising:

-   -   a 5′-non translated region (NTR) comprising the sequence ACCAGC        (SEQ ID NO. 8) at, or proximal to, its 5′-terminus;    -   a HCV polyprotein coding region; and    -   a 3′-NTR region.

In a second embodiment, the present invention is directed to a HCVself-replicating polynucleotide encoding a polyprotein comprising one ormore amino acid substitution selected from the group consisting of:R(1135)K; S(1148)G; S(1560)G; K(1691)R; L(1701)F; I(1984)V; T(1993)A;G(2042)C; G(2042)R; S(2404)P; L(2155)P; P(2166)L and M(2992)T.

Particularly, the invention is directed to a HCV self-replicatingpolynucleotide encoding a polyprotein comprising the any one of theamino acid substitutions as described above, further comprising theamino acid substitution E(1202)G.

Alternatively, the first embodiment of the present invention is directedto HCV self-replicating polynucleotide molecule comprising a G2042C/Rmutation.

According to the second embodiment, the present invention particularlyprovides a HCV polynucleotide construct comprising:

-   -   a 5′-NTR region comprising the sequence ACCAGC (SEQ ID NO. 8)        at, or proximal to, its 5′-terminus;    -   a HCV polyprotein region coding for a HCV polyprotein comprising        a G(2042)C or a G(2042)R mutation; and    -   a 3′-NTR region.

Preferably, the polynucleotide construct of the present invention is aDNA or RNA molecule. More preferably, the construct is a RNA molecule.Most preferably, the construct is a DNA molecule.

More particularly, the first embodiment of this invention is directed toa RNA molecule encoded by the DNA molecule selected from the groupconsisting of: SEQ ID NO. 2, 4, 5, 6, 7, 24 and 25.

Most particularly, the invention provides a DNA molecule selected fromthe group consisting of: SEQ ID NO. 2, 4, 5, 6, 7, 24 and 25.

In a third embodiment, the invention also is directed to an expressionvector comprising DNA forms of the above polynucleotide, operably linkedwith a promoter.

Preferably, the promoter is selected from the group consisting of: T3,T7 and SP6.

According to a fourth embodiment, there is provided a host celltransfected with the self-replicating polynucleotide or vector asdescribed above. Particularly, the host cell is a eukaryotic cell line.More particularly, the eukaryotic cell line is a hepatic cell line. Mostparticularly, the hepatic cell line is Huh-7.

In a fifth embodiment, the present invention provides a RNA replicationassay comprising the steps of:

-   -   a) incubating the host cell as described above under conditions        suitable for RNA replication;    -   b) isolating the total cellular RNA from the cells; and    -   c) analyzing the RNA so as to measure the amount of HCV RNA        replicated.

Preferably, the analysis of RNA levels in step c) is carried out byamplifying the RNA by real-time RT-PCR analysis using HCV specificprimers so as to measure the amount of HCV RNA replicated.

Alternatively in this fifth embodiment, the construct comprises areporter gene, and the analysis of RNA levels in step c) is carried outby assessing the level of reporter expressed.

According to a preferred aspect of the sixth embodiment, the inventionis directed to a method for testing a compound for inhibiting HCVreplication, including the steps of:

-   -   a) carrying step a) as described in the above assay, in the        presence or absence of the compound;    -   b) isolating the total cellular RNA from the cells; and    -   c) analyzing the RNA so as to measure the amount of HCV RNA        replicated.    -   d) comparing the levels of HCV RNA in cells in the absence and        presence of the inhibitor,        wherein reduced RNA levels is indicative of the ability of the        compound to inhibit replication.

Preferably, the cell line is incubated with the test compound for about3-4 days at a temperature of about 37° C.

EXAMPLES Example 1 Replicon Constructs APGK-12; FIG. 1

pET9a-EMCV was obtained by ligating an oligonucleotide linker 5′gaattccagatggcgcgcccagatgttaaccagatccatggcacactctagagtactgtcgac 3′ (SEQID NO.9) to pET-9a (Novagen) that was cut with EcoRI and SalI to formthe vector pET-9a-mod. This linker contains the following restrictionsites: EcoRI, AscI, HpaI, NcoI, XbaI, ScaI, SalI. The EMCV IRES wasamplified by PCR from the vector pTM1 with primers 5′cggaatcgttaacagaccacaacggtttccctc 3′ (SEQ ID NO.10) and 5′ggcgtacccatggtattatcgtgtttttca 3′ (SEQ ID NO.11) and ligated intopET-9a-mod via EcoRI and NcoI to form pET-9a-EMCV.

The sequence of HCV NS2 to NS5B followed by the 3′UTR of HCV wasobtained from the replicon construct I377/NS2-3′ (Lohman et al., 1999Science 285:110-113; accession number: AJ242651) and synthesized byOperon Technologies Inc. with a T to C change at the NcoI site in NS5Bat nucleotide 8032. This sequence was released from an GenOp® vector(Operon Technologies) with NcoI and ScaI and transferred intopET-9a-EMCV to form pET-9a-EMCV-NS2-5B-3′UTR.

pET-9a-HCV-neo was obtained by amplification of the HCV IRES from a HCVcDNA isolated from patient serum with primers 5′gcatatgaattctaatacgactcactataggccagcccccgattg 3′ (SEQ ID NO.12)containing a T7 promoter and primer 5′ggcgcgccctttggtttttctttgaggtttaggattcgtgctcat 3′ (SEQ ID NO.13) andamplification of the neomycin phosphotransferase gene from the vectorpcDNA 3.1 (Invitrogen) with primers 5′aaagggcgcatgattgaacaagatggattgcacgca 3′ (SEQ ID NO.14) and 5′gcatatgttaactcagaagaactcgtcaagaaggcgata 3′ (SEQ ID NO.15). These two PCRfragments were mixed and amplified with primers 5′gcatatgaaftctaatacgactcactataggccagcccccgattg 3′ (SEQ ID NO.16) and 5′gcatatgttaactcagaagaactcgtcaagaaggcgata 3′ (SEQ ID NO.15), cut with EcoRI and HpaI and transferred into pET-9a-mod to form pet-9a-HCV-neo. TheEMCV-NS2-5B-3′UTR was released from pET-9a-EMCV-NS2-5B-3′UTR with HpaIand ScaI and transferred into pet-9a-HCV-neo that was cut with HpaI toform pET-9a-APGK12. This insert was sequenced with specific successiveprimers using a ABI Prism® BigDye™ Terminator Cycle sequencing kit andanalyzed on ABI Prism® 377 DNA Sequencer and is shown in SEQ ID NO 1.

RNA In Vitro Transcription

pET-9a-APGK12 DNA was cut with ScaI for expression of the full-lengthreplicon or with BgIII for expression of a truncated negative controlRNA. DNA was analyzed on a 1% agarose gel and purified byPhenol/Chloroform extraction. RNA was produced using a T7 Ribomax® kit(Promega) followed by extraction with phenol/chloroform andprecipitation with 7.5 M LiCl₂. RNA was treated with DNAse I for 15 minto remove the DNA template and further purified with an RNeasy® column(Qiagen). RNA integrity was verified on a denaturing formaldehyde 1%agarose gel.

Example 2 Primary Transfection of Huh7 Cells and Selection of RepliconCell Lines

Human hepatoma Huh7 cells (Health Science Research Resources Bank,Osaka, Japan) were grown in 10% FBS/DMEM. Cells were grown to 70%confluency, trypsinized, washed with phosphate buffered saline (PBS) andadjusted to 1×10⁷ cells/ml of PBS. 800 μl of cells were transferred into0.4 cm cuvettes and mixed with 15 μg of replicon RNA. Cells wereelectroporated using 960 μF, 300 volts for ˜18 msec and evenlydistributed into two 15 cm tissue culture plates and incubated in atissue culture incubator for 24 hours. The selection of first and secondgeneration replicon cell lines was with 10% FBS/DMEM medium supplementedwith 1 mg/ml of G418. Cells were selected for 3-5 weeks until colonieswere observed that were isolated and expanded.

Following the G418 selection and propagation of Huh-7 cells transfectedwith APGK12 (SEQ ID NO. 1) RNA, cells that formed a distinct colony weretreated with trypsin and serially passed into larger culture flasks toestablish cell lines. Approximately 10×10⁶ cells were harvested fromeach cell line. The cells were lysed and the total cellular RNAextracted and purified as outlined in Qiagen RNAeasy® preparatoryprocedures. FIG. 2 shows the analysis of 12 μg of total cellular RNAfrom various cell lines as analyzed on a Northern blot of a denaturingagarose-formaldehyde gel.

FIG. 2A is a Northern blot (radioactively probed with HCV specificminus-strand RNA) that detects the presence of plus-strand replicon RNA.Lanes 1 and 2 are positive controls that contain 10⁹ copies of in vitrotranscribed APGK12 RNA. Lane 2 contains the in vitro transcribed RNAmixed with 12 μg of total cellular from naïve Huh-7 cells. Lane 3 is anegative control of total cellular RNA from untreated Huh-7 cells. Lanes4 and 5 contain cellular RNA from the B1 and B3 G418 resistant celllines that have DNA integrated copies of the neomycin phosphotransferasegene. Lane 6 contains total cellular RNA from a Huh-7 cell line,designated S22.3, that harbors high copy number of HCV sub-genomicreplicon RNA as detected by the positive signal in the 8 kilo-baserange. Other cell lines have no detectable replicon RNA. FIG. 2B is aNorthern blot of a duplicate of the gel presented in 2A with theexception that the blot was radioactively probed with HCV specificplus-strand RNA to detect the presence of HCV minus-strand RNA (lanes 1and 2 are positive control lanes that contain 10⁹ copies of full lengthgenomic HCV minus strand RNA); only lane 6, which contains 12 μg oftotal cellular RNA from cell line S22.3, harbors detectable minus-strandreplicon RNA at the expected size of 8-9 kilobases. An quantitativeestimation of RNA copy number, based on phosphorimager scanning of theNorthern blots, is approximately 6×10⁷ copies of plus-strand/μg of totalRNA, and 6×10⁶ copies of minus strand/μg of total RNA. The presence ofthe plus-strand and minus-strand intermediate confirms that the HCVsub-genomic RNA is actively replicating in the S22.3 cell line.

Example 3 S22.3 Cell Line Constitutively Expresses HCV Non-StructuralProteins

HCV non-structural protein expression was examined in the S22.3 cellline. FIG. 3 displays the result of indirect immunofluorescence thatdetects the HCV NS4A protein in the S22.3 cell line and not in thereplicon negative B1 cell line (a G418 resistant Huh-7 cell line).Indirect immunofluorescence was performed on cells that were culturedand fixed (with 4% paraformaldehyde) onto Lab-tek chamber slides. Cellswere permeabilized with 0.2% Triton X-100 for 10 minutes followed by a 1hour treatment with 5% milk powder dissolved in phosphate-bufferedsaline (PBS). A rabbit serum containing polyclonal antibody raisedagainst a peptide spanning the HCV NS4A region was the primary antibodyused in detection. Following a 2 hour incubation with the primaryantibody, cells were washed with PBS and a secondary goat anti-rabbitantibody conjugated with red-fluor Alexa® 594 (Molecular Probes) wasadded to cells for 3 hours. Unbound secondary antibody was removed withPBS washes and cells were sealed with a cover slip. FIG. 3 (top panels)shows the results of immunofluorescence as detected by a microscope withspecific fluorescent filtering; the bottom panels represent theidentical field of cells viewed by diffractive interference contrast(DIC) microscopy. The majority of S22.3 (FIG. 3A) cells within the fieldstain positively for HCV NS4A protein that localizes in the cytoplasm,whereas the B1 cells (FIG. 3B) that fail to express any HCV proteins,only have background level of staining. A small proportion of S22.3cells express high levels of intensely stained HCV NS4A.

Expression of the proteins encoded by the bi-cistronic replicon RNA wasalso examined on Western-blots following SDS-PAGE separation of totalproteins extracted from: (i) naïve Huh-7 cell line, (ii) neomycinresistant Huh-7 cell line B1, and (iii) the S22.3 cell line. FIG. 4panels A, B, and C, demonstrate the results of western blots probed withrabbit polyclonal antisera specific for neomycin phosphotransferase(NPT), HCV NS3, and HCV NS5B, respectively. Visualization was achievedthrough autoradiographic detection of a chemiluminescent reactivesecondary HRP-conjugated goat anti-rabbit antibody. FIG. 4 panel A showsthat the S22.3 RNA replicon cell line, expresses the NPT protein atlevels higher than B1 cells (which contain an integrated DNA copy of thenpt gene) and that the naïve Huh-7 cell line does not produce the NPTprotein. FIG. 4 panels B and C show that only the S22.3 cell lineproduces the mature HCV NS3 and NS5B proteins, respectively. The westernblots demonstrate that the S22.3 cell line, which harbors activelyreplicating HCV sub-genomic replicon RNA, maintains replication of theRNA through the high level expression of the HCV non-structuralproteins.

Example 4 Sequence Determination of Adapted Replicons

Total RNA was extracted from replicon containing Huh7 cells using aRNeasy Kit (Qiagen). Replicon RNA was reverse transcribed and amplifiedby PCR using a OneStep RT-PCR kit (Qiagen) and HCV specific primers (asselected from the full-length sequence disclosed in WO 00/66623). Tendistinct RT-PCR products, that covered the entire bi-cistronic repliconin a staggered fashion, were amplified using oligonucleotide primers.The PCR fragments were sequenced directly with ABI Prism® BigDye™Terminator Cycle PCR Sequencing and analyzed on ABI Prism® 377 DNASequencer. To analyze the sequence of the HCV replicon 3′ and 5′ ends aRNA ligation/RT-PCR procedure described in Kolykhalov et al. 1996 J. ofVirology, 7, p. 3363-3371 was followed. The nucleotide sequence of S22.3is presented as SEQ ID NO. 2.

Example 5 Serial Passage of HCV Replicon RNA

The total cellular RNA from the S22.3 cell line was prepared asdescribed above. HCV Replicon RNA copy number was determined by Taqman®RT-PCR analysis and 20 μg of total S22.3 cellular RNA (containing 1×10⁹copies of HCV RNA) was transfected by electroporation into 8×10⁶ naïveHuh-7 cells. Transfected cells were subsequently cultured in 10 cmtissue culture plates containing DMEM supplemented with 10% fetal calfserum (10% FCS). Media was changed to DMEM (10% FCS) supplemented with 1mg/ml G418 24 hours after transfection and then changed every threedays. Twenty-three visible colonies formed three to four weekspost-transfection and G418 selection. G418 resistant colonies wereexpanded into second generation cell lines that represent the first celllines harboring serially passaged HCV Replicon RNA. Three of these celllines: R3, R7, and R16 were the subject of further analyses. First, theefficiency of transduction by each of the adapted replicons wasdetermined by electroporation of the total cellular RNA (extracted fromthe R3, R7 and R16) into naïve Huh-7 cells; following electroporation,the transduction efficiency was determined as described above, bycounting the visible G418 resistant colonies that arose following 3 to 5weeks of G418 selection (Table 1). Second, the sequence of the seriallypassed adapted replicons was determined from the total cellular RNA thatwas extracted from each of the R3, R7 and R16 replicon cell lines asdescribed in example 4 (SEQ ID NO. 4, 5, 6). Using the pAPGK12 as areference sequence (SEQ ID NO. 1), the nucleotide changes that wereselected in HCV segment of the adapted replicons are presented in FIG.5A. Some of these nucleotide changes are silent and do not change theencoded amino acid whereas others result in an amino acid substitution.FIG. 5B summarizes the amino acid changes encoded by the adaptedreplicons with the amino acid sequence of pAPGK12 as the reference. Itis important to note that the reference sequence APGK-12 (SEQ ID NO. 1)contains an extra G at the 5′-terminal (5′-GG) that is not maintained inthe replicating RNA of the established cell lines. Also noteworthy isthat, in addition to G→A at nucleotide 1, there is also an adaptedmutation G→C/R at amino acid 2042 (shown as amino acid 1233 in thesequence listing since a.a. 810 of NS2 is numbered as a.a. 1 in SEQ ID)that can be found in all clones analyzed.

TABLE 1 Transfection of Huh-7 cells RNA Copies of Replicon # ColoniesSEQ ID 5 ng APKG12 replicon 1.2 × 10⁹   0 in 20 μg total Huh-7 RNA 15 μgAPKG12   3 × 10¹² 1 (S22.3) 1 replicon RNA 20 μg total: S22.3 cellularRNA   3 × 10⁹ 23 (3 clones 2 analyzed) R3 cellular RNA   1 × 10⁹  200 4R7 cellular RNA   1 × 10⁹  20 5 R16 cellular RNA   3 × 10⁸  100 6 clonedR3rep RNA 2.3 × 10⁸ 2000 7

Example 6 Construction of APGK12 with 5′G→A Substitution (APGK12-5′A,SEQ ID NO.24)

The pAPGK12 DNA was modified to change the first nucleotide in thesequence to replace the 5′GG with a 5′A. The change in the pAPGK12 wasintroduced by replacing an EcoRI/AgeI portion of the sequence with aPCR-generated EcoRI/AgeI fragment that includes the mutation. Theoligonucleotides used for the amplification were (SEQ ID. NO. 20):5′-GTG GAC GAA TTC TAA TAC GAC TCA CTA TAA CCA GCC CCC GAT TGG-3′ and(SEQ ID. NO. 21): 5′-GGA ACG CCC GTC GTG GCC AGC CAC GAT-3′ andgenerated a 195 bp DNA fragment that was then digested with EcoRI andAgeI. The resulting 178 bp restriction fragment was used to replace theEcoRI/AgeI fragment in pAPGK12 to generate the pAPGK12-5′A plasmid.

Example 7 cDNA Cloning of the R3-Replicon (R3rep)

The cDNA clone of the R3 replicon was produced by RT-PCR of RNAextracted from the R3 cell line. The following two oligonucleotides wereused: (SEQ ID. NO. 22): 5′-GTC GTC TTC TCT GAC ATG GAG AC-3′ and (SEQID. NO. 23): 5′-GAG TTG CTC AGT GGA TTG ATG GGC AGC-3′. The ˜4400 nt PCRfragment, starting within the NS2 coding region and extending to the5′-end of the NS5B coding region, was cloned into the plasmid pCR3.1 byTA cloning (Invitrogen). The SacII/XhoI portion of this R3 sequence wasthen used to replace the SacII/XhoI fragment present in the pAPGK12 andthe pAPGK12-5′A described above. Consequently, two R3 cDNA sequenceswere generated: (I) R3-Rep-5′G with an initiating 5′G (SEQ ID NO.7), andR3-Rep-5′A (SEQ ID NO.25) with an initiating 5′A. Sequencing of the R3rep cDNA identified unique nucleotide changes that differ from theoriginal pAPGK12 sequence (see FIG. 5A); some of these changes aresilent and do not change the encoded amino acid, whereas others doresult in an amino acid change (see FIG. 5B). The differences between R3and the R3-rep reflect the isolation of a unique R3-rep cDNA cloneencoding nucleotide changes that were not observed from the sequencingof the total RNA extracted from the R3 cell line.

Example 8 Efficiency of Colony Formation with Modified Constructs

RNA from pAPGK12, pAPGK12-5′A, pR3-Rep and pR3-Rep-5′A was generated byin vitro transcription using the T7 Ribomax® kit (Promega) as describedin example 1 above. The reactions containing the pAPGK12-5′A andpR3-Rep-5′A templates were scaled-up 10-fold due to the limitation ofcommercial RNA polymerase in initiating transcripts with 5′-A. The fulllength RNAs and control truncated RNA for each clone were introducedinto 8×10⁶ naïve Huh-7 cells by electroporation as described in example2. Replicon RNA was supplemented with total cellular Huh-7 carrier RNAto achieve a final 15-20 μg quantity. The cells were then cultured inDMEM medium supplemented with 10% fetal calf serum and 0.25 mg/ml G418in two 150 mm plates. The lower concentration of G418 was sufficient toisolate and select replicon containing cell lines as none of thetransfectants with the control truncated RNA produced any resistantcolonies. In contrast, in vitro transcribed APGK-12 RNAs that harboreither a 5′G or 5′A form colonies with the same efficiency (ca. 80cfu/μg in FIG. 6 panels A and B) following selection with G418. Variousquantities (ranging from 0.1 ng to 1 μg) of the R3-rep-5′A RNA, weretransfected into naïve Huh-7 cells to determine a colony formationefficiency of 1.2×10⁶ cfu/μg of RNA (FIG. 6 panel C depicts transfectionwith 1 μg of RNA). Various quantities (ranging from 0.1 ng to 1 μg) ofR3-rep [5′G] were similarly transfected resulting in a colony formationefficiency of 2×10⁶ cfu/μg of RNA (FIG. 6 panel D depicts colonyformation with 1 μg of RNA). Note that, shown for the first time, HCVsubgenomic replicons replicate as efficiently with a 5′A nucleotide inplace of the 5′G. APGK12 with a 5′A or 5′G RNA have similar transductionefficiencies. Similarly, R3-Rep RNAs with either the 5′A or 5′G bothdisplay the markedly increased transduction efficiency. Notably, theadaptive mutants within the HCV non-structural segment encoded by theR3-Rep provides for a substantial increase in transduction efficiency asdepicted by the dramatic increase in colony forming units per μg oftransfected RNA.

Example 9 Quantification of HCV Replicon RNA Levels in Cell Lines

S22.3 cells, or cell lines harboring other adapted replicons, wereseeded in DMEM supplemented with 10% FBS, PenStrep and 1 μg/mLGeneticin. At the end of the incubation period the replicon copy numberis evaluated by real-time RT-PCR with the ABI Prism 7700 SequenceDetection System. The TAQMAN® EZ RT-PCR kit provides a system for thedetection and analysis of HCV RNA (as first demonstrated by Martell etal. 1999 J. Clin. Microbiol. 37: 327-332). Direct detection of thereverse transcription polymerase chain reaction (RT-PCR) product with nodownstream processing is accomplished by monitoring the increase influorescence of a dye-labeled DNA probe (FIG. 6). The nucleotidesequence of both primers (adapted from Ruster, B. Zeuzem, S. and Roth,W. K., 1995. Analytical Biochemistry 224:597-600) and probe (adaptedfrom Hohne, M., Roeske, H. and Schreier, E. 1998, Poster Presentation:P297 at the Fifth International Meeting on Hepatitis C Virus and RelatedViruses Molecular Virology and Pathogenesis, Venezia-Lido Italy, Jun.25-28, 1998) located in the 5′-region of the HCV genome are thefollowing:

HCV Forward primer: (SEQ ID NO.17) 5′ ACG CAG AAA GCG TCT AGC CAT GGCGTT AGT 3′ HCV Reverse primer: (SEQ ID NO.18) 5′ TCC CGG GGC ACT CGC AAGCAC CCT ATC AGG 3′ HCV Probe: (SEQ ID NO.19) 5′ FAM-TGG TCT GCG GAA CGGGTG AGT ACA CC-TAMRA 3′ FAM: Fluorescence reporter dye. TAMRA: Quencherdye.

Using The TAQMAN® EZ RT-PCR kit, the following reaction was set up:

Volume per sample Final Component (μL) Concentration RNase-Free Water 16— 5X Taqman EZ Buffer 10 1X Manganese Acetate 25 mM 6 3 mM dATP 10 mM1.5 300 μM dCTP 10 mM 1.5 300 μM dGTP 10 mM 1.5 300 μM dUTP 20 mM 1.5300 μM HCV Forward Primer 10 μM 1 200 nM HCV Reverse Primer 10 μM 1 200nM HCV Probe 5 μM 2 200 nM rTth DNA Polymerase 2 0.1 U/μL 2.5 U/μLAmpErase UNG 1 U/μL 0.5 0.01 U/μL Total Mix 45 —

To this reaction mix, 5 μL of total RNA extracted from S22.3 cellsdiluted at 10 ng/μL was added, for a total of 50 ng of RNA per reaction.The replicon copy number was evaluated with a standard curve made fromknown amounts of replicon copies (supplemented with 50 ng of wild typeHuh-7 RNA) and assayed in an identical reaction mix (FIG. 7).

Thermal cycler parameters used for the RT-PCR reaction on the ABI Prism7700 Sequence Detection System were optimized for HCV detection:

Temperature Time Cycle (° C.) (Minutes) Repeat Reaction Hold 50 2Initial Step Hold 60 30  Reverse Transcription Hold 95 5 UNGDeactivation Cycle 95 0:15 2 Melt 60 1 Anneal/Extend Cycle 90 0:15 40Melt 60 1 Anneal/Extend

Quantification is based on the threshold cycle, where the amplificationplot crosses a defined fluorescence threshold. Comparison of thethreshold cycles provides a highly sensitive measure of relativetemplate concentration in different samples. Monitoring during earlycycles, when PCR fidelity is at its highest, provides precise data foraccurate quantification. The relative template concentration can beconverted to RNA copy numbers by employing a standard curve of HCV RNAwith known copy number (FIG. 7).

Example 10 A Specific HCV NS3 Protease Anti-Viral Compound InhibitsReplication of the HCV Replicon in S22.3 Cell Lines

In order to determine the effect of a specific HCV NS3 proteaseanti-viral compound on replicon levels in S22.3 cells, the cells wereseeded in 24 Well Cell Culture Cluster at 5×10⁴ cells per well in 500 μLof DMEM complemented with 10% FBS, PenStrep and 1 μg/mL Geneticin. Cellswere incubated until compound addition in a 5% CO₂ incubator at 37° C.The dose-response curve of the inhibitor displayed 11 concentrationsresulting from serial two-fold dilutions (1:1). The startingconcentration of compound A was 100 nM. One control well (without anycompound) was also included in the course of the experiment. The 24 wellplates were incubated for 4 days in a 5% CO₂ incubator at 37° C.Following a 4 day incubation period, the cells were washed once with PBSand RNA was extracted with the RNeasy® Mini Kit and Qiashredder® fromQiagen. RNA from each well was eluted in 50 uL of H₂O. The RNA wasquantified by optical density at 260 nm on a Cary 1E UV-VisibleSpectrophotometer. 50 ng of RNA from each well was used to quantify theHCV replicon RNA copy number as detailed in Example 6. The level ofinhibition (% inhibition) of each well containing inhibitor wascalculated with the following equation (CN=HCV Replicon copy number):

${\% \cdot {inhibition}} = {\left( \frac{{{CN} \cdot {control}} - {{CN} \cdot {well}}}{{CN} \cdot {control}} \right)*100}$

The calculated % inhibition values were then used to determine IC₅₀,slope factor (n) and maximum inhibition (I_(max)) by the non-linearregression routine NLIN procedure of SAS using the following equation:

${\% \cdot {inhibition}} = \frac{I_{\max} \times \lbrack{inhibitor}\rbrack^{n}}{\lbrack{inhibitor}\rbrack^{n} + {IC}_{50}^{n}}$

Compound A was tested in the assay at least 4 times. The IC₅₀ curveswere analyzed individually by the SAS nonlinear regression analysis.FIG. 8 shows a typical curve and Table 2 shows the individual andaverage IC₅₀ values of compound A. The average IC₅₀ of compound A in thereplication assay was 1.1 nM.

TABLE 2 IC₅₀ of compound A in the S22.3 Cell line Replicon Assay.Compound IC₅₀ (nM) Average IC₅₀ (nM) A 1.2 1.2 1.0 0.9 1.1 ± 0.2Discussion

The reproducible and robust ex vivo propagation of hepatitis C virus, tolevels required for the accurate testing of potential anti-viralcompounds, has not been achieved with any system. As an alternativeapproach to studying the molecular mechanisms of hepatitis C virus RNAreplication, selectable self-replicating bi-cistronic RNAs weredeveloped (Lohman et al., 1999, Science 285, 110-113; Bartenschlager, R.et al., 1993, J. Virol., 67, 3835-3844 CA 2,303,526). Minimally, thesereplicons encode for some or all of the non-structural proteins and alsocarry a selectable marker such as the neomycin phosphotransferase.Though intracellular steady-state levels of these sub-genomic repliconRNAs among the selected clones is moderate to high, the frequency ofgenerating G418-resistant colonies upon transfection of the consensusRNA described by Lohman et al. or Bartenschlager, R. et al., 1993, J.Virol., 67, 3835-3844 is very low. Less than 100 colonies are generatedwhen 8 million cells are transfected with 1 μg of in vitro transcribedbi-cistronic replicon RNA. A low efficiency of colony formation wasfirst noted by Lohmann et al (1999 et al, Science 285, 110-113). Sincethen, Lohmann et al. (2001) J. Virol. 1437-1449, Blight et al. 2000,Science 290, 1972-1974, and Guo et al., (2001) J. Virol. 8516-8523, haveisolated sub-genomic RNAs with markedly improved efficiencies in thecolony formation assay. Lohmann et al., 1999 Science 285, 110-113originally reported that selection of sub genomic replicons may notinvolve the selection of adaptive mutants as serially passaged RNA didnot demonstrate an improved transfection efficiency. Nevertheless, in aneffort to characterize the function and fitness of replicating HCV RNA,we serially passaged the replicon RNA that was isolated from the firstselected cell-line. Notably, a significant increase in colony formingefficiency was obtained from this experiment, even though the quantityof replicon RNA was orders of magnitude lower than originally used totransfect the in vitro transcribed RNA. Furthermore, a second roundserial passage of replicon RNA from this first generation clone intonaive Huh-7 cells provided for yet another increase in colony formationefficiency (Table 1).

Our analysis of replicating HCV RNAs identified several adaptivemutations that enhance the efficiency of colony formation by up to 4orders of magnitude. Adaptive mutations were found in manynon-structural proteins, as well as in the 5′ non-translated region. Thesubstitution of the 5′-GG doublet for a 5′-A as the inauguratingnucleotide of the HCV 5′-UTR is a variant of the HCV genome that has notbeen previously described, despite the sequencing of innumerablegenotypes and subtypes from across the world. Our original replicon thatcarried a 5′-GG evolved to variants with either a single 5′-A or 5′-G,both of which showed equal transduction efficiency. We describe here thefirst report of a HCV genome that can tolerate and stably maintain a 5′Aextremity. Moreover, we were successful in re-introducing this definedsingle nucleotide substitution into our cDNA clone and generate in vitrotranscribed RNA harboring such an extremity to confirm that a 5′Afunctions as efficiently as a 5′G.

We have identified adaptive amino acid substitutions in the HCVnon-structural proteins NS3, NS4A and NS5A in the R3 replicon, and asubstitution in NS5B in the R7 clone (see FIG. 5B). These mutations,particularly the combination defined by the R3-rep (SEQ ID NO. 7), whenreconstituted into a cDNA clone and transcribed onto a RNA replicon,result in a significantly enhanced transduction efficiency of up to20,000 fold from the original wild type APGK12 replicon RNA. However,the steady state levels of intracellular replicon RNA were comparablefrom each of the different isolated clones. This result suggests thatthe increase in replication efficiency by the adaptive mutations doesnot result in higher stable intracellular RNA levels due to higher RNAreplication, but rather confers increased permissivity for establishingthe replicon in a greater number of Huh7 cells. Such a phenotype may bemanifested transiently, through an initial increase of the amount of denovo replication, that is required to surpass a defined threshold toestablish persistently replicating RNAs within a population of dividingcells.

Recently three other groups also identified other distinct adaptivemutants. Lohmann et al. (2000) reported enhanced transductionefficiencies of up to 10,000 fold with mutations in NS3, NS4B, NS5A andNS5B. Blight et al. 2000, Science 290:1972-1974 reported an augmentationof transduction efficiencies up to 20,000 fold with a single mutation inNS5A whereas Guo et al., (2001) J. Virol. 8516-8523 reported increasesin transduction efficiencies of 5,000-10,000 fold with a deletion of asingle amino acid in NS5A. The amino acid substitutions that we describehere have not previously been identified as adaptive mutants thatenhance the efficiency of RNA transfection and/or replication. Oneexception is the mutation of E1202G in NS3 that we found in both the R7and R16 replicons. This adaptation was previously described by Guo etal., (2001) J. Virol. 8516-8523 and Krieger et al (2001) J. Virol.4614-4624. All other adaptive mutations, without exception, describedherein are unpublished.

The development of selectable subgenomic HCV replicons has provided forpotential avenues of exploration on HCV RNA replication, persistence,and pathogenesis in cultured cells. However, the low transductionefficiency with the HCV RNA-containing replicons as originally described(Lohmann et al., 1999 Science 285: 110-113) showed that it was not apractical system for reverse genetics studies. The adaptive mutantsdescribed herein overcome the low transduction efficiency. In light ofthe recent descriptions of adaptive mutants by other groups, we notethat adaptation can be achieved by distinct mutations in different HCVNS proteins, although the level of adaptation can vary drastically. Thereplicons encoding adaptive mutants that are described herein areideally suited for reverse genetic studies to identify novel HCV targetsor host cell targets that may modulate HCV RNA replication or HCVreplicon RNA colony formation. The adapted and highly efficientreplicons are suitable tools for characterizing subtle genotypic orphenotypic changes that affect an easily quantifiable transductionefficiency.

Lastly, we have used our adapted HCV sub genomic replicon cell-line todemonstrate the proficient inhibition of HCV RNA replication by aspecific small molecule inhibitor of the HCV NS3 protease. This is thefirst demonstration that an antiviral, designed to specifically inhibitone of the HCV non-structural proteins, inhibits HCV RNA replication incell culture. Moreover, this compound and our S22.3 cell line validatethe proposal that RNA replication is directed by the HCV non-structuralproteins NS3 to NS5B. The assay that we have described and validatedwill be extremely useful in characterizing other inhibitors of HCVnon-structural protein function in cell culture in a high throughputfashion.

All references found throughout the present disclosure are hereinincorporated by reference whether they be found in the following list ornot.

1. An isolated host cell transfected with a self-replicatingpolynucleotide comprising: (a) a 5′-Non Translated Region; (b) a HCVpolynucleotide coding region encoding an HCV polyprotein comprising:NS3, NS4A, NS4B, NS5A, and NS5B proteins, said polynucleotide codingregion comprising SEQ ID NO. 4 or SEQ ID NO. 7, and (c) a 3′-NonTranslated Region.
 2. The host cell according to claim 1, wherein thehost cell is a eukaryotic cell line.
 3. The host cell according to claim2, wherein said eukaryotic cell line is a hepatic cell line.
 4. The hostcell according to claim 3, wherein said hepatic cell line is Huh-7. 5.The isolated host cell transfected with a self-replicatingpolynucleotide according to claim 1, wherein the 5′-Non TranslatedRegion is ACCAGC (SEQ ID No. 8).
 6. The isolated host cell transfectedwith a self-replicating polynucleotide according to claim 1, wherein the5′-Non Translated Region is GCCAGC (SEQ ID No. 27).
 7. A RNA replicationassay comprising the steps of: (a) incubating the host cell according toclaim 1 under conditions suitable for RNA replication; (b) isolating thetotal cellular RNA from the cells; and (c) analyzing the RNA so as tomeasure the amount of HCV RNA replicated.
 8. The assay according toclaim 7, wherein the analysis of RNA levels in step (c) is carried outby amplifying the RNA by real-time RT-PCR analysis using HCV specificprimers so as to measure the amount of HCV RNA replicated.
 9. The assayaccording to claim 7, wherein said polynucleotide encodes for a reportergene, and the analysis of RNA levels in step (c) is carried out byassessing the level of reporter expressed.
 10. A method for testing acompound for inhibiting HCV replication, including the steps of: (a)incubating the host cell according to claim 1 under conditions suitablefor RNA replication in the presence or absence of the compound; (b)isolating the total cellular RNA from the cells; (c) analyzing the RNAso as to measure the amount of HCV RNA replicated; and (d) comparing thelevels of HCV RNA in cells in the absence and presence of the inhibitor,wherein reduced RNA levels is indicative of the ability of the compoundto inhibit replication.
 11. The method according to claim 10, whereinsaid cell is incubated with the test compound for about 3-4 days at atemperature of about 37° C.